G protein-coupled receptors (GPCRs) control key second messengers such as Ca , phosphoinositides (IP) and cAMP. Upon the interaction with activated GPCR, the G protein binds GTP and dissociates into the Ga-GTP and the tightly associated GBy dimer, which modulate the activity their target effector enzymes or ion channels. While these basic steps are established, mechanisms of regulation of these pathways with respect to specificity, kinetics and crosstalk between distinct G protein circuits, are not understood. This research program stems from the Pi's earlier discovery showing that neuronal cells contain a novel kind of G protein heterodimer - the complex of GB5 and RGS proteins. RGS proteins are a large family of regulators of G protein signaling that act as GTPase activating proteins (GAPs) for Ga subunits. Only one subfamily of RGS proteins, those containing a Gy-like (GGL) domain, interacts with GB5, and this project is focused on GB5-RGS7 complex, which is widely expressed in the brain. Studies in this and other labs showed that GB5 and RGS7 are always associated in native tissues. Each subunit of the GB5-RGS7 complex rapidly degrades in the absence of the other, and so in GPS knock-out mice, RGS7 is absent. In reconstituted systems, GB5-RGS7 can attenuate Gi-, and Gq-mediated signals but underlying molecular mechanisms are poorly understood and it is not known if both Gi and Gq pathways are regulated in vivo. In addition, unexpected preliminary data of this proposal identified Gs as a potential binding partner of the GB5-RGS7 complex. Surprisingly, the interaction with Gs involved not the RGS domain of RGS7 but its unique DEP domain. This proposal will use biochemical, cellular and genetic strategies to further unravel the role of GB5-RGS7 in signal transduction. Specific Aim 1 will investigate the mechanism of inhibition of Gq signaling by GB5-RGS7 using reconstitution with Gq-coupled GPCRs in transfected model cells, and measuring kinetics of Ca2+ release and IP production. Specific Aim 2 will explore the interaction between GB5-RGS7 and Gas by a series of protein-protein interaction assays and analysis of pathways regulating cAMP production. Specific Aim 3 will use mice lacking GB5- RGS complex to compare signal transduction patterns of primary cultured neurons obtained from GB5-/- mice with wild-type. In all, this multidisciplinary program will achieve the following goals: (i) establish which G protein pathways are regulated by GB5-RGS7 in native cells, and study how this regulation occurs at the molecular level; (ii) test the hypothesis that GB5-RGS7 can act in receptor-selective manner. Brain GPCRs are a major current and future therapeutic target, and this research will lead to new insights into the mechanisms that regulate signaling from these receptors.